Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures

Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures

FEMS Microbiology Ecology 41 (2002) 151^160 www.fems-microbiology.org Nitrate is reduced by heterotrophic bacteria but not transferred to Prochloroc...

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FEMS Microbiology Ecology 41 (2002) 151^160

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Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures Antonio Lo¤pez-Lozano, Jesu¤s Diez, Sabah El Alaoui, Conrado Moreno-Vivia¤n, Jose Manuel Garc|¤a-Ferna¤ndez  Departamento de Bioqu|¤mica y Biolog|¤a Molecular, Edi¢cio Severo Ochoa, 1a planta, Campus de Rabanales, Universidad de Co¤rdoba, E-14071 Co¤rdoba, Spain Received 13 March 2002 ; received in revised form 24 April 2002; accepted 26 April 2002 First published online 10 July 2002

Abstract The ability to assimilate nitrate in non-axenic isolates of Prochlorococcus spp. was addressed in this work, particularly in three lowirradiance adapted strains originating from ocean depths with measurable nitrate concentrations. None of the studied strains was able to use nitrate as the sole nitrogen source. Nitrate reductase (NR; EC 1.6.6.2) activity was, however, detected using the methyl viologen/ dithionite assay in crude extracts from all studied Prochlorococcus strains. Characterization of this activity unambiguously demonstrated its enzymatic origin. We observed that NR activity did not decrease in vivo under darkness. Attempts to detect the narB gene (coding for NR in other cyanobacteria) by PCR with primers designed on the basis of the specific codon usage in Prochlorococcus were unsuccessful. However, when primers were designed considering the codon frequencies typical of other bacteria, we could amplify different fragments of nas genes, coding for bacterial assimilatory NRs. Similar amplification products were obtained using colonies of contaminant bacteria from Prochlorococcus cultures as PCR template. Furthermore, NR activity was found in cultures of these contaminants, demonstrating the non-cyanobacterial origin of the enzyme. These results strongly suggest that the studied strains of Prochlorococcus lack NR, in spite of inhabiting environments with nitrate as the main nitrogen source. In addition, they indicate that the nitrite produced by heterotrophic bacteria is not transferred to Prochlorococcus for growth, thus discarding a trophic nitrogen chain between heterotrophic bacteria and Prochlorococcus in the studied cultures. ; 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Prochlorococcus ; Cyanobacterium ; Nitrogen assimilation; Nitrate reductase; Marine phytoplankton

1. Introduction Prochlorococcus is a marine oxyphotobacterium [1^3], ubiquitous and abundant in most oligotrophic regions of the world’s oceans. Its importance in terms of global primary production, together with several unusual features with regard to other cyanobacteria, has led to an increasing number of studies on this microorganism (for a review, see [4]). Among these studies, only a few have dealt with nutrient assimilation [3,5^9], and in particular nitrogen assimilation remains largely unknown. However, it is known that nitrogen is one of the main limiting nutrients

* Corresponding author. Tel. : +34 (957) 211 075; Fax : +34 (957) 218 592. E-mail address : [email protected] (J.M. Garc|¤a-Ferna¤ndez). Abbreviations : NR, nitrate reductase ; PCC, Pasteur Culture Collection; RCC, Rosco¡ Culture Collection

in marine ecosystems, e.g. in the North Atlantic [10]. Since Prochlorococcus populations are particularly abundant in very oligotrophic regions of the oceans, with barely detectable levels of nitrogen [4], the elucidation of the nitrogen assimilatory pathway in Prochlorococcus appears to be one of the key topics to understanding its ecological success. We have previously studied the physiological regulation of glutamine synthetase [5,6] in the axenic, high-light adapted strain Prochlorococcus PCC 9511 [3], in order to avoid confusing results due to the contaminant bacteria in the other Prochlorococcus strains described to date [4]. This strain shows a striking feature: it is unable to grow on nitrate as the sole nitrogen source [3,5], contrary to most cyanobacteria, living either in freshwater or marine environments [11^13]. There are only four reported genera of cyanobacteria that include strains unable to utilize nitrate, and it has been suggested that the presence of environmental ammonium could allow the loss of this ability [14]. However, the inability to assimilate nitrate seems an

0168-6496 / 02 / $22.00 ; 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 1 6 8 - 6 4 9 6 ( 0 2 ) 0 0 2 9 7 - 0

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Table 1 Prochlorococcus strains used in this work (obtained from the Rosco¡ Culture Collection) Strain

Other name

Origin

Depth (m)

Light adaptation

SS120 (RCC 156) NATL1 (RCC 277) NATL2 (RCC 280) MED4 (RCC 1543) TAK9803-2 (RCC 264)

CCMP 1375 FP12 FP5 CCMP 1378

Sargasso Sea North Atlantic North Atlantic NW Mediterranean Paci¢c Ocean

120 30 30 5 5

Low irradiance Low irradiance Low irradiance High irradiance High irradiance

ecological contradiction in the low-light adapted Prochlorococcus strains that grow at depths where ammonium concentration is non-detectable and nitrate is likely the main nitrogen source [15]. Furthermore, it has been demonstrated that nitrate addition has a stimulating e¡ect on the cell cycle of Prochlorococcus in the Mediterranean Sea [16], and the median cell depth for Prochlorococcus is positively correlated with the nitracline in the North Atlantic [17]. Consequently, we decided to address the ability for nitrate utilization in Prochlorococcus spp. with the following strategy: ¢rst, we tested the possibility of growth on nitrate in various non-axenic Prochlorococcus strains, including three low-light adapted strains from the Rosco¡ Culture Collection (RCC); second, we used cell extracts from these cultures to detect and characterize a possible nitrate reductase (NR) activity, and to test whether it belongs to Prochlorococcus or to contaminant bacteria; and third, we looked for NR genes using conserved motifs in the assimilatory NRs from cyanobacteria and heterotrophic bacteria to design speci¢c primers for PCR ampli¢cation, considering either the preferential codon usage of Prochlorococcus or the codon frequencies of other bacteria. Our attention was specially focused on the low-light adapted Prochlorococcus strains due to the presence of detectable levels of nitrate in their natural environment; since the only axenic Prochlorococcus strains available to date are high-light adapted [3], we had to use non-axenic low-light adapted Prochlorococcus strains in this study, although de¢nitive conclusions could only be reached once some of these strains are made axenic.

provided by Carlos Masso¤ de Ariza (Instituto Espan‹ol de Oceanograf|¤a, Spain). Cells were grown in a culture room set at 24‡C under continuous blue light (photon £ux densities of 4 and 40 WE m32 s31 ). All experiments were performed during the exponential phase of growth. Growth was determined by measuring the optical density of cultures at 674 nm. 2.2. In vivo experiments Cultures (250-ml aliquots) were harvested by centrifugation at the indicated times at 30 100Ug for 5 min in an Avanti J-25 Beckman centrifuge equipped with a JA-14 rotor. After pouring o¡ most of the supernatant and carefully pipetting out the remaining medium, the pellet was directly resuspended in 500 Wl of cold 50 mM Tris^HCl, pH 7.5, and immediately frozen at 320‡C until use for enzymatic analysis. In the experiments following the growth of Prochlorococcus on ammonium vs. nitrate (Fig. 1), standard cultures initially growing on 400 WM (NH4 )2 SO4 were divided into two aliquots; then these were replenished either with standard PCR-S11 medium (control with ammonium) or with PCR-S11 where ammonium was replaced by KNO3 at a ¢nal concentration of 800 WM. In the experiments designed to study the e¡ect of transfer to nitrate (Table 2), cells were collected by centrifugation as indicated above, washed with N-free PCR-S11 medium, and resuspended in new medium containing the indicated concentrations of (NH4 )2 SO4 or KNO3 . For experiments requiring darkness, culture bottles were completely covered with two layers of aluminum foil, and sampling was performed in the dark.

2. Materials and methods 2.3. Enzymatic assays 2.1. Prochlorococcus strains and culturing The Prochlorococcus strains studied in the present work (Table 1) are non-axenic, and correspond to two groups: ecotypes adapted to low irradiance, SS120 (RCC 156), NATL1 (RCC 277), NATL2 (RCC 280), and to high irradiance, MED4 (RCC 1543) and TAK9803-2 (RCC 264). They were obtained from the Rosco¡ Culture Collection and were routinely cultured in polycarbonate Nalgene £asks (10 l) using PCR-S11 medium as described by Rippka and coworkers [3]. The seawater used as a basis for this medium was obtained from the Mediterranean Sea, kindly

After thawing, the samples were directly used for NR assay without further treatment, since previous studies demonstrated that no additional disruption of cells is needed to assay enzymes of Prochlorococcus [5]. NR activity was determined basically as previously described [18]. The reaction mixture contained (in order of addition): 450 Wl of distilled water; 200 Wl of 0.5 M phosphate bu¡er, pH 7.0; 100 Wl of 0.2 M KNO3 ; 100 Wl of 40 mM methyl viologen; 50 Wl of cell extract; and 100 Wl of 0.1 M Na2 S2 O4 (prepared in 0.3 M NaHCO3 ). The blank reaction was stopped at t = 0 by vigorous vortex mixing (to

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oxidize the viologen). After 30 min at 37‡C, the remaining reactions were stopped by the same method. 50 Wl of 2 M ZnSO4 and 50 Wl of 2 M NaOH were added to each mixture, to remove the green debris that could interfere with the spectrophotometric determinations [19], and the tubes were centrifuged at 16 000Ug for 5 min. The supernatant was transferred to clean tubes, and then 1 ml of 58 mM sulfanilamide (4-aminobenzenesulfonamide) and 1 ml of 0.77 mM NNEDA (N-(1-naphthyl)ethylenediamine) were added to each mixture to determine the formation of nitrite. After 10 min to allow the appearance of the pink color, absorbance at 540 nm was determined in a Beckman DU-640 spectrophotometer, to calculate nitrite concentration [20]. One unit of NR activity is the amount of enzyme that catalyzes the formation of 1 Wmol of nitrite per minute. Total protein concentration of cell extracts was determined in triplicate using the Bio-Rad protein assay, following the instructions of the manufacturer, and used to standardize the enzymatic activities. In assays to study the optimum pH of the NR activity, the reaction was prepared with an equimolar mixture (at a ¢nal concentration of 0.1 M) of bu¡ers (Tris^HCl, K2 HPO4 and citric acid) with di¡erent pKa values, allowing coverage of the pH range from 4 to 10. 2.4. PCR ampli¢cation, DNA sequencing and sequence analysis The deduced amino acid sequences from narB genes available at GenBank in March 1999 : Nostoc sp. strain PCC 7120 (PID 1071640), Synechococcus sp. strain PCC 7942 (PID 397157), Synechocystis sp. strain PCC 6803 (PID 1652567) and Oscillatoria chalybea (PID 899356), were aligned with the software MegAlign 4.0, from the package LaserGene Navigator (DNAStar Inc.). The motifs CPYCGVGC and PNAMGGRE (positions 14^24 and 346^353 of the NarB protein from Synechocystis sp. strain PCC 6803, respectively), fully conserved in the four genes, were selected to design narB-speci¢c primers using the software Oligo v 4.05 (National Biosciences, Plymouth, UK), taking into account the preferential use of codons by Prochlorococcus, as described [4] : FNR24P : 5P-TGYCCWTATTGYGGWGTWGGWTGY-3P; RNR24P: 5PYTCWCGWCCWCCCATWGCATTWGG-3P. The corresponding fully degenerate primers were also tested. Table 2 NR activities in samples of several Prochlorococcus strains after 24 h of transfer to the indicated nitrogen sources Nitrogen source

400 WM (NH4 )2 SO4 800 WM KNO2 800 WM KNO3

NR activity (mU mg31 ) SS120

MED4

TAK9803-2

1.33 X 0.01 1.83 X 0.15 5.92 X 0.36

1.43 X 0.15 1.58 X 0.18 4.40 X 0.58

0.87 X 0.07 0.89 X 0.24 1.57 X 0.16

Data are mean values of three independent determinations X S.D.

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PCR was performed using 25 pmol of each primer, concentrations of 50 WM for each deoxynucleoside triphosphate, 2.5 U of AmpliTaq Gold (Perkin-Elmer) and 1UTaq bu¡er containing 3 mM MgCl2 . The PCR ampli¢cation protocol was as follows : 5 min at 94‡C; 35 cycles of: 60 s at 94‡C ; 60 s at 60‡C ; 90 s at 72‡C; and ¢nally 15 min at 72‡C. PCR was performed in a Perkin-Elmer 2400 thermocycler, using 200-Wl tubes. Pelleted cells from a Prochlorococcus culture (2 ml), harvested at the exponential phase of growth, were centrifuged at 13 000Ug for 5 min and used as template for the PCR. Alternatively, genomic DNA obtained according to the method described by Cai and Wolk [21] was utilized as PCR template. In addition, a wider range of assimilatory NR sequences from heterotrophic bacteria and cyanobacteria were used to design nas-speci¢c primers : Klebsiella pneumoniae (PID 541199), Klebsiella oxytoca (PID 4755082), Bacillus subtilis (PID 2828506), Shewanella frigidimarina (PID 2275099), O. chalybea, Synechocystis sp. strain PCC 6803 and Synechococcus sp. strain PCC 7942 (cyanobacterial PIDs indicated above); in this case, the motifs TGQPNAMGGRE and GTMTNSERRI (positions 343^353 and 466^ 475 of the NarB protein from Synechocystis sp. strain PCC 6803, respectively) were selected, and the codon usage was biased towards that of high G+C content phototrophic or heterotrophic bacteria : NAS-2 : 5P-ACSGGSCAGCCSAACGCSATGGGSGSCCGSGA-3P; NAS-3 : 5P-GAYSCGSCGYTCCGAGTTSGTCAYSGTGCC-3P. PCR was performed using 10 pmol of each primer, concentrations of 50 WM for each deoxynucleoside triphosphate, 5 U of AmpliTaq Gold (Perkin-Elmer) and 1UTaq bu¡er containing 3 mM MgCl2 . The PCR ampli¢cation protocol was as follows : 5 min at 96‡C; 10 cycles of: 1 min at 94‡C; 1 min at 70‡C ; then 30 cycles of: 30 s at 94‡C; 30 s at 89‡C ; 1 min at 70‡C; and ¢nally 5 min at 70‡C. The rest of the conditions were the same as above. The PCR products were directly sequenced at the Servicio Centralizado de Apoyo a la Investigacio¤n (Universidad de Co¤rdoba, Spain) using an ABI PRISM 373 sequencer and a Big Dye Terminator kit, following the instructions of the manufacturer. Analysis of the PCR products was performed by translated blast searches (blast-x) at http://www.ncbi.nlm.nih. gov/BLAST/. Alignment of the deduced protein sequences and phylogenetic trees was obtained using the Clustal method with MegAlign 4.0 (DNAStar Inc). 2.5. Prochlorococcus genomic sequences Preliminary sequence data on the Prochlorococcus MED4 and MIT 9313 genomes were obtained from the DOE-Joint Genome Institute (JGI) at http://www.jgi.doe. gov/JGI_microbial/html/prochlorococcus_med4/prochlo_ med4_homepage.html and http://www.jgi.doe.gov/JGI_ microbial/html/prochlorococcus_mit9313/prochlo_mit9313 _homepage.html/.

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Fig. 1. Growth of ¢ve Prochlorococcus strains (SS120, NATL1, NATL2, MED4, TAK9803-2) after transfer from ammonium- to nitrate-containing PCR-S11 medium. Arrows indicate the times of medium replenishment : b, cultures growing on ammonium (control); F, cultures transferred to nitrate.

3. Results 3.1. Transfer of cells from ammonium- to nitrate-containing medium The ability to grow on nitrate as the sole nitrogen source was assessed in ¢ve strains of Prochlorococcus spp., including representatives of the three main phylogenetic clusters described to date: MED4, the extensively studied high-light ecotype, corresponds to the high-light I (HL-I) clade and TAK9803-2, another high-light adapted strain, corresponds to the high-light II (HL-II) clade (a clade clearly de¢ned by phylogenetic analysis using 16S rRNA gene sequences; [22]). Finally, we also selected for this study three low-light (LL) adapted strains: SS120 ^ the model LL ecotype, NATL1 and NATL2, as we assumed that these ecotypes could possess NR, being abundant at depths where nitrate is probably the main nitrogen source. Prochlorococcus cultures of all ¢ve strains, exponentially growing in standard PCR-S11 (i.e., with ammonium as the sole nitrogen source), were divided into two aliquots of 25 ml ; one of them was diluted at 0 time with 1 vol. (25 ml) of standard PCR-S11, while the other was diluted with 1 vol. of nitrate-containing PCR-S11. The growth of cultures was determined during the following weeks (Fig. 1), performing a medium replenishment (either with ammonium- or nitrate-containing PCR-S11 medium when appropriate, indicated in the ¢gure with an arrow) to ensure that growth was not limited by other nutrients. In all cases, the cells transferred to nitrate were unable to grow under such conditions, while those on ammonium

were growing at standard rates. It is particularly interesting to note the similar behavior of all the studied Prochlorococcus strains, regardless of their phylogenetic relationship or adaptation to either high or low irradiance. Supplementation of Na2 MoO4 (20 WM ¢nal concentration) to the medium containing nitrate, in order to ensure that the molybdenum concentration was not limiting for the cells to synthesize the molybdenum cofactor of NR, had no e¡ect on the growth of cultures (not shown). 3.2. Nitrate reductase activity assays on Prochlorococcus cell extracts Since none of the available Prochlorococcus strains was found to grow on nitrate, we studied the possibility of NR occurrence in cell extracts. Initial attempts were done to determine the activity of this enzyme, which can be measured with the system methyl viologen/sodium dithionite as the electron donor, using standard concentrations of all reagents as previously described [18]. However, no positive results were found. Increasing the methyl viologen concentration from 1.5 to 4 mM, and of potassium nitrate from 10 to 20 mM in the reaction mixture, allowed detection of activity of ca. 1 mU mg31 protein in cell extracts obtained from standard ammonium-growing cultures (Table 2). When the assay was performed on cells which had been transferred to nitrate for 24 h, the NR activity increased more than 3-fold (for MED4 cultures) or 4-fold (for SS120 cultures), up to values of 5.92 mU mg31 protein. In similar transfer experiments of both strains to nitrite, the level of NR activity was not signi¢cantly a¡ected. Preliminary characterization of the NR activity was car-

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increase in nitrite concentration is catalyzed by an enzyme, and is not the result of non-biological reactions. 3.3. E¡ect of darkness on nitrate reductase activity in Prochlorococcus cultures Since NR is regulated by light in most photosynthetic organisms, we studied the e¡ect of transferring the cultures from light to darkness on the NR levels. Prochlorococcus SS120 cells growing on ammonium at 4 WE m32 s31 were centrifuged, washed and transferred to PCR-S11 containing nitrate. Then the culture was divided into two aliquots; one was kept under the standard irradiance, and the other was subjected to darkness for 8 h. We followed the time course in NR activity. Fig. 3 shows the results from this experiment : the speci¢c NR showed no clear changes when comparing results from samples subjected to light or darkness, showing similar values at the end of the experiment. These results indicate that the NR found in cell extracts was apparently una¡ected by the photosynthetic activity of Prochlorococcus. Nitrite concentration measured in the culture media (which oscillated between 0.3 and 0.9 WM) did not increase after nitrate addition (not shown), showing similar values to those detected in PCRS11 medium. Fig. 2. Preliminary characterization of the nitrate reductase activity from Prochlorococcus SS120 cell extracts. A: E¡ect of the pH of the reaction mixture. B: E¡ect of the volume of cell extract used per assay. C: E¡ect of the time of assay on the nitrite produced. Data are mean values of three independent determinations X S.D.

ried out in order to con¢rm its enzymatic origin. We observed that v 90% of total NR activity was found in the pellet of thawed cell extracts, after centrifugation during 5 min at 13 100Ug (not shown). Treatment of cells with mixed alkyltrimethylammonium bromide (a permeabilizing agent widely used in enzymatic studies on cyanobacteria) during 1 or 2 min induced an almost complete loss of nitrate reductase activity (not shown). Consequently it was not used for the preparation of cell extracts. Instead, the resuspended cells were used directly for enzymatic assays, and the cell debris was removed after the assays as described in Section 2, to avoid interference during the spectrophotometric determinations of absorbance. The NR activity completely disappeared when cell extracts were boiled for 5 min prior to the enzymatic assay (not shown). Replacement of methyl viologen/sodium dithionite by NADH as the electron donor led to a complete loss of enzymatic activity (not shown). The optimum pH of the NR assay was 7.0 (Fig. 2A), with a decrease in the NR activity of ca. 50% at pH 10. Besides, we observed that the determined values of nitrite showed very good linear correlation with the volume of cell extract used for the assay (Fig. 2B) and also with the time of assay (Fig. 2C). All this information indicates that the observed

3.4. Use of PCR for detection of narB genes in Prochlorococcus spp. We tried to detect the possible presence of narB gene (encoding NR in other cyanobacteria) in cultures of Prochlorococcus spp. PCR ampli¢cation was performed using either whole cells or genomic DNA from Prochlorococcus MED4, TAK9803-2, NATL1, NATL2 and SS120 cultures as template, using narB-speci¢c primers (FNR24P and RNR24P) designed according to the codon usage of Prochlorococcus, as described in Section 2. Di¡erent ampli¢cation conditions were tested (i.e., a range of magnesium chloride concentrations, polymerases, annealing temperatures, etc.; not shown). In all cases, no positive results were obtained with these sets of primers, suggesting the absence of narB in the studied strains.

Fig. 3. Time course of NR in Prochlorococcus SS120 cultures transferred to nitrate. a, light, nitrate ; b, darkness, nitrate. Data are mean values of three independent determinations X S.D.

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Fig. 4. A: Alignment of the deduced amino acid sequences from PCR products obtained using nas-designed primers and pelleted cells from Prochlorococcus SS120, NATL1, MED4 and TAK9803-2 cultures as template for PCR ampli¢cation. The contaminant sequence corresponds to the PCR product obtained from a plated colony of heterotrophic bacteria (see Section 3). B: Phylogenetic tree of prokaryotic NRs, including all available complete cyanobacterial NarB sequences at GenBank (as of October 10, 2001) together with 10 representative sequences from heterotrophic bacteria. Accession numbers are indicated in Section 2.

However, when nas-speci¢c primers (NAS-2 and NAS3) designed taking into account the codon usage of high G+C content phototrophic and heterotrophic bacteria were used, we were able to amplify DNA fragments of ca. 360 bp in all strains. This size agrees with the length of the gene region located between both primers in the di¡erent nas genes. Sequencing of such fragments showed that they corresponded to nas sequences coding for putative assimilatory NRs (Fig. 4A), showing a certain degree of variability among the di¡erent isolates. In order to check whether such PCR products were derived from Prochlorococcus or from contaminants, we plated out samples of Prochlorococcus SS120 liquid culture media onto Petri

dishes containing PCR-S11 medium with 0.8% agar and 1 mM glucose to allow heterotrophic growth. After 24 h at 37‡C to allow bacterial growth, we performed PCR ampli¢cations utilizing the nas-speci¢c primers and cells from a single colony as PCR template, as described above. The PCR products were again of about 360 bp and closely matched those obtained from pelleted cells from Prochlorococcus cultures (Fig. 4A). Interestingly, the sequence of the PCR product from that colony (derived from a SS120 culture) was more similar to those obtained from NATL1 or TAK9803-2 than to the PCR product obtained from SS120 (Fig. 4B); this suggests the presence of more than one bacterium producing NR in the Prochlorococcus

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SS120 cultures. The G+C content in the PCR products ranged from 56% (TAK9803-2) to 64% (MED4), clearly di¡erent from the values described for Prochlorococcus genomes (50.7% in MIT 9313, and 30.8% in MED4 [23]). All these sequences grouped with NRs from heterotrophic bacteria in a phylogenetic tree including all available cyanobacterial narB sequences and 10 representative sequences from heterotrophic bacteria (Fig. 4B), showing identity values higher than 50% with sequences from Agrobacterium tumefaciens, Mesorhizobium loti or Caulobacter crescentus, while identities with the cyanobacterial counterparts were in all cases lower than 40%. These results strongly suggest the lack of narB in the studied strains of Prochlorococcus. Further con¢rmation of the contaminant origin of the observed NR was obtained by culturing the plated contaminants. Bacterial cells from the above mentioned plate were grown for several days on liquid PCR-S11 medium containing 1 mM glucose and either 400 WM (NH4 )2 SO4 (control) or 800 WM KNO3 . Cells were subjected to the same treatment as standard Prochlorococcus samples (centrifugation, freezing/unfreezing, resuspension in 50 mM Tris^HCl pH 7.5 bu¡er, etc.) and the obtained extract was assayed. We observed that NR occurred in both kinds of culture ; the activity also appeared in the pellet of samples after centrifugation (not shown), and it was increased in the nitrate-containing samples when compared with control, ammonium-containing samples (not shown); these results are consistent with those shown in Table 2.

4. Discussion Nitrate utilization by phytoplankton is a central topic in oceanography, due to its consequences on primary production and carbon £uxes [15,24^26]. Given the importance of Prochlorococcus in the phytoplankton populations, especially in oligotrophic regions, and the poor understanding of nutrient utilization in this oxyphotobacterium [4], we decided to use di¡erent biochemical and molecular approaches, both in vivo and in vitro, to study the ability to assimilate nitrate in various Prochlorococcus strains. There is no Prochlorococcus strain thus far reported to grow on nitrate as the sole nitrogen source. Yet, nitrate assimilation is a feature found in all studied groups of cyanobacteria, even those able to perform nitrogen ¢xation [13]. In the only axenic Prochlorococcus strain described to date, the high-light adapted PCC 9511, the inability to utilize nitrate has been demonstrated [3,5]. This ¢nding has been reinforced by the availability of the full sequence of the MED4 genome (http://www.jgi.doe. gov/JGI_microbial/html/prochlorococcus_med4/prochlo_ med4_homepage.html) which is most probably identical to that of PCC 9511 [3], where the genes narB and nirA

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(coding for nitrate and nitrite reductases, respectively) are lacking [27]. Our initial hypothesis was based on the knowledge of typical nitrate pro¢les in the oceans, where signi¢cant nitrate concentrations are usually detected at or near the deep chlorophyll maximum [4]. Since Prochlorococcus populations are abundant at such depths, we expected that the low-irradiance adapted strains of Prochlorococcus could include some representatives able to utilize nitrate. We show in the present work that, contrary to our expectations, none of the studied strains could grow after transfer from ammonium- to nitrate-containing medium (Fig. 1), in spite of medium replenishments to ensure good supply of other nutrients. However, Prochlorococcus cultures are quite di⁄cult to maintain in good shape, and it could be argued that the results from this experiment do not completely discard the ability of nitrate assimilation in Prochlorococcus. Consequently, we utilized additional approaches to check our hypothesis. NR enzymatic assays were performed on cell extracts from Prochlorococcus SS120, the model low-irradiance strain, and the other strains, ¢nding signi¢cant NR activity (Table 2). We con¢rmed by di¡erent techniques (Fig. 2) that this activity e¡ectively corresponded to an enzyme and is not the result of an artifact. However, since the studied Prochlorococcus strains are non-axenic, it was necessary to ¢nd methods allowing this activity to be assigned either to the heterotrophic contaminant bacteria or to Prochlorococcus. The optimum pH of the NR activity in Prochlorococcus SS120 was 7.0 (Fig. 2), in sharp contrast to that described for other NRs from cyanobacteria, e.g. Synechococcus PCC 7942 or Plectonema boryanum, which have a pH optimum of ca. 10 [28,29]. This fact suggests that the NR detected in Prochlorococcus extracts could be produced by contaminants. The NR activity was found in the pellet of unfrozen samples after centrifugation; this observation could suggest either that the enzyme is linked to membranes (typical of assimilatory NRs from cyanobacteria), or that the cells are not broken by the freezing/thawing protocol; we have previously demonstrated that this protocol permeabilizes the Prochlorococcus cells, allowing most of the glutamine synthetase to be detected in the supernatant [5]. However, many heterotrophic bacteria are not permeabilized in this way, and consequently the second possibility cannot be discounted if the origin of the NR activity is from contaminant bacteria. Nitrate reduction is a genuine photosynthetic process in cyanobacteria [12,13], and consequently it is upregulated by light [28,30^33]. Therefore, we analyzed the e¡ect of darkness on the NR activity found in Prochlorococcus SS120 cultures in short term experiments (Fig. 3), as the response of NR to changes in light is quick in cyanobacteria [30]. We observed no clear e¡ect of the transfer from light to darkness on the NR activities, as could be ex-

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pected if the enzyme was produced by a photosynthetic organism; this reinforces its belonging to contaminant non-photosynthetic bacteria, whose NR activity would be light-independent. Finally, PCR ampli¢cation using narB-designed primers did not permit the ampli¢cation of narB fragments in any of the tested strains, regardless of the level of degeneration considered for the codons. However, nas-designed primers did permit the ampli¢cation of partial fragments of nas genes from all studied isolates, suggesting that contaminants were producing the observed NR. The e¡ectiveness of these primers has been demonstrated previously by ampli¢cation of fragments of the assimilatory NR genes from Rhodobacter capsulatus E1F1 and the Gram-positive bacterium Rhodococcus sp. RB1 (C. Pino, F. Olmo-Mira, M.D. Rolda¤n, F. Castillo and C. Moreno-Vivia¤n, unpublished). The ¢nding of very similar nas PCR products from plated contaminant colonies (Fig. 4), and the detection of NR activity in cultures of such contaminant colonies, demonstrate the presence in Prochlorococcus cultures of contaminant heterotrophic bacteria that possess nas genes and express an active NR; furthermore, this activity was higher when cells were transferred to nitrate-containing PCR-S11 medium, in agreement with the results observed using Prochlorococcus cultures (Table 2). However, it is di⁄cult to correlate these results with the available information on Prochlorococcus abundance and the concentration of nitrogen in the oceans [34]. Although high Prochlorococcus concentrations have been reported over a range of nitrate concentrations of 4 orders of magnitude, with usually the peak of Prochlorococcus abundance located above the nitracline [34], addition of nitrate has been shown to stimulate the rate of cell cycling in Prochlorococcus in the Mediterranean Sea [16], and Prochlorococcus abundance increased after nitrogen enrichment in the North Atlantic [35]. It has been suggested that the available nitrate could be converted to another utilizable source of nitrogen for Prochlorococcus by other constituents of the natural microbial community, allowing its growth in spite of its inability to use nitrate [3]. However, our results do not support this hypothesis, since the contaminant bacteria seem to be able to utilize nitrate, increasing their NR activities speci¢cally after transfer to nitrate (Table 2), but we found no growth at all in the coexistent Prochlorococcus populations (Fig. 1). This could be due to the non-excretion of reduced forms of nitrogen by the contaminant bacteria (as suggested by the almost constant nitrite concentration observed in our cultures), indicating that they utilize all the reduced nitrite. Consequently, no transfer of nitrogen from bacteria to Prochlorococcus was detected in our experiments. While this is a noteworthy observation, the overall situation could be very di¡erent in nature, as most marine heterotrophic bacterial populations are non-culturable to date [36], and thus it is unclear how representative our samples are from the

natural composition of the heterotrophic bacteria in the oceans. An alternative explanation would derive from the high genetic diversity of Prochlorococcus [37^40], that could include some ecotypes able to assimilate nitrate. The natural vertical gradients of nutrients have been proposed as one of the driving forces inducing the speciation within the genus Prochlorococcus [4]. This hypothesis seems particularly well suited to the case of nitrogen sources, as reduced forms are found in the surface of the oceans, while at depth oxidized forms become predominant. The reported inability to use urea in the marine Synechococcus strain WH 7803, while 20 other tested strains could use this nitrogen source [41], could be another example of speciation promoted by the di¡erent nitrogen sources availability in marine ecosystems. Nevertheless, a recent report suggests the possibility of amino acid-like molecules available at depth in the oceans [42] ; if this idea holds true, it could explain why organisms unable to use nitrate can survive in such environments, assimilating the amine group from reduced forms of nitrogen through the glutamine synthetase/glutamate synthase cycle.

5. Concluding remarks If we consider our results together with the available information of nitrogen utilization by Prochlorococcus, a clear outcome is that Prochlorococcus seems to be the ¢rst group of Oxyphotobacteria lacking the ability to grow on nitrate, a feature that has for a long time been considered one of the basic features of cyanobacterial metabolism [11^13]. This fact represents a key di¡erence with most known marine Synechococcus strains [27,41,43,44], and could be due to evolutionary pressures to remove genes non-essential for proliferation in oligotrophic environments, where usually Prochlorococcus outcompetes Synechococcus [34]. Hence, the e¡ects of nutrient availability (in particular nitrogen) could play a larger role on the phylogenetic diversi¢cation of phytoplankton than previously expected. In this sense, it is very interesting to note that the genome of the Prochlorococcus strain MIT 9313 (isolated at 135 m depth in the North Atlantic) contains a gene with signi¢cant homology with cyanobacterial nirA, encoding nitrite reductase, while the genes for nitrate uptake and reduction are missing [27]; furthermore, it has been reported that MIT 9313 and three other Prochlorococcus strains are able to grow on nitrite [27,38]. An interesting topic for future research is to analyze the reasons why cooccurring cyanobacteria such as the marine Synechococcus group have kept the genes for nitrate assimilation [27], and to determine the relevance of this on the di¡erent distributions [34] and physiologies [45] of both organisms.

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Acknowledgements This work was supported by the EU (program MASTIII, project PROMOLEC, MAS3-CT97-0128), the Junta de Andaluc|¤a, Spain (II Plan Andaluz de Investigacio¤n, CVI 0123) and the University of Co¤rdoba, Spain (Programa Propio de Investigacio¤n de la UCO). A.L.-L. received a fellowship from CVI 0123 (Junta de Andalucia). S.E.A. received a doctoral fellowship from the Agencia Espan‹ola de Cooperacio¤n Internacional (AECI), Spain. J.M.G.-F. was recipient of post-doctoral grants from the EU (TMR and MASTIII programs) and of a return grant from the Spanish Ministerio de Educacio¤n y Ciencia. Work of C.M.-V. was supported by grants from Junta de Andaluc|¤a (CVI 0117) and CICYT (PB98 1022 CO2 01).We thank Dr. F. Partensky (Station Biologique de Rosco¡, France) and Dr. D.J. Scanlan (University of Warwick, UK) for providing Prochlorococcus strains and helpful discussions; Dr. W.R. Hess (Humboldt Universita«t zu Berlin, Germany), Dr. R. Rippka (Institut Pasteur, France) and Dr. L.R. Moore (University of Maine, USA) for stimulating discussions; and Carlos Masso¤ de Ariza, the Centro Oceanogra¤¢co de Fuengirola (Ma¤laga, Spain) and the Instituto Espan‹ol de Oceanograf|¤a for kindly organizing the supply of seawater.

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